Brain dopamine is postulated to modulate alcohol consumption. We hypothesize that reduction in extracellular dopamine (DA) levels would reduce ethanol intake. The main goal of this proposal is to examine if transplantation of neural stem cells (NSCs) modified to overexpress the human DA transporter (hDAT) into specific brain sites can reduce extracellular DA levels and alter ethanol?s behavioral actions. NSCs are pluripotent cells that exist in the developing and adult brain. NSCs have a capacity to differentiate into all known neural type cells including neurons, astrocytes and oligodendrocytes. Unprecedented plasticity of NSCs makes them ideal candidates for genetic modification and transplantation into the central nervous system (CNS). This proposal will explore a novel strategy using stem cell transplantation for moderating alcohol effects and intake. The specific aims of this proposal are: (1) Generation of neural stem cell lines expressing human DAT (hDAT) under control of an inducible promoter. Because cell lines expressing high levels of hDAT suitable for neural transplantation do not exist, we propose to generate a hDAT-expressing high neural stem cell line (C17.hDAT) for cell transplantation. We will use mouse v-myc immortalized neural stem cells (C17.2) obtained from Dr. Evan Snyder as the cell line development platform. (2) Examine the hDAT expression and function in C17.hDAT stem cell-derived neurons and glia. We propose to examine hDAT expression and function in stem cells differentiated into neurons or glia. This aim will also examine the effectiveness of regulating Tet-On system-driven hDAT expression and function with doxycycline. (3) Determine if transplantation of C17.hDAT stem cells into mouse brain can reduce extracellular dopamine in vivo and alter ethanol?s actions. We will first transplant C17.hDAT cells developed in the aim 1 into the brains of wildtype (wt) and DAT knockout mice provided by Dr. Marc Caron and into the brains of wt mice with high (C57BL/6) and low (DBA) alcohol preference. Target areas for grafting will include nucleus accumbens, prefrontal cortex, amygdala and dorsal striatum. Grafting into DAT knockout mice should reverse the behavioral and biochemical consequences of reduction in DAT levels. Survival of grafted cells and function of transgenic hDAT will be examined. Behavioral testing will examine ethanol-induced locomotor activity and the mice will be exported to other sites in the consortium to test for alcohol preference. These studies will generate hDAT-expressing stem cells for transplantation in the CNS and examine whether stem cell therapy can reduce alcohol?s actions. Such findings will provide the first evidence for use of stem cells in the treatment of drug dependence.